Structural mechanisms of Clostridium difficile pathogenesis

 描述（由申请方提供）：艰难梭菌是一种革兰氏阳性、孢子形成厌氧菌，可感染结肠，引起一系列人类疾病，包括腹泻、伪膜性结肠炎和毒性巨结肠。与C.艰难梭菌感染（CDI）的增加，使C.这是一个重大的公共卫生问题。C.艰难梭菌的致病机制是TcdA和TcdB，这两种大的同源毒素能够进入并修饰真核宿主细胞内的多个靶标。该提案旨在跟进上一个资助周期中的两个重要发现，TcdA的晶体结构和TcdB的细胞受体。在目标1中，我们将使用TcdA递送结构域的结构作为理解宿主内体膜内毒素孔形成的框架。电子显微镜、交联和荧光强度测量的混合方法将产生毒素孔的结构视图。在目标2中，我们将使用电子显微镜、诱变、定量结合研究和X射线晶体学的混合物来定义TcdB与其受体PVRL 3的相互作用。在目的3中，我们将评估TcdB-PVRL 3相互作用在中毒和感染的小鼠模型中的相关性。从这些目标的结果将提供一个分子的理解，允许毒素进入宿主细胞和治疗干预的框架的事件。

 DESCRIPTION (provided by applicant): Clostridium difficile is a gram-positive, spore-forming anaerobe that infects the colon, causing a range of human disease including diarrhea, pseudomembranous colitis, and toxic megacolon. The incidence, severity, and costs associated with C. difficile infection (CDI) are increasing, making C. difficile a significant public health concern. The principle virulence factors in C. difficile pathogenesis are TcdA and TcdB, two large homologous toxins capable of entering and modifying multiple targets within eukaryotic host cells. This proposal is designed to follow up two important discoveries made in the previous funding cycle, a crystal structure of TcdA and the cellular receptor for TcdB. In Aim 1, we will use the structure of the TcdA delivery domain as a framework for understanding toxin pore formation within the host endosomal membrane. A hybrid approach of electron microscopy, crosslinking, and fluorescence intensity measurements will generate a structural view of the toxin pore. In Aim 2, we will define the interaction of TcdB with its receptor, PVRL3, using a hybrid of electron microscopy, mutagenesis, quantitative binding studies, and X-ray crystallography. In Aim 3, we will evaluate the relevance of the TcdB-PVRL3 interaction in mouse models of intoxication and infection. Results from these aims will provide a molecular understanding of the events that allow toxins access to the host cell and a framework for therapeutic intervention.